1. Field of the Invention
This invention relates to a novel thrombin-binding substance, and, more particularly, to a thrombin-binding substance useful as a medicine, especially a medicine for curing thrombosis and the like, involving anticoagulation and fibrinolytic systems which control blood coagulation, and a process for the preparation of the thrombin-binding substance.
2. Description of the Background
A great deal of work have been done regarding the role that thrombin plays as a proteolytic enzyme in the control mechanism of blood coagulation and the mechanism of blood coagulation has been elucidated for the most part.
According to a publication it was reported that thrombin activated Protein C which is said to act on the fibrinolytic and anticoagulant systems. The publication also reports that there is a certain substance in extracts of rabbit lung tissue which functions as a coenzyme for the activation mechanism. Such a substance was named thrombomodulin [N. L. Esmon et al, J. Biological Chemistry, 257, (2), 859-864 (1982)].
N. Aoki, one of the present inventors, and others have reported human thrombomodulin separated from human placenta and having characteristics similar to the thrombomodulin reported by J. Biological Chemistry, with a molecular weight of about 71,000 under unreduced conditions [Thromb. Res., 37, 353-364 (1985)].
Furthermore, I. Maruyama et al reported that the comparison of activities of human thrombomodulin separated from human placenta with a molecular weight of about 71,000 (under unreduced conditions) and those of the above-mentioned rabbit thrombomodulin revealed that they had identical activities [J. Clin. Invest., 75, 987-991 (1985)].
It has also been reported by H. Ishii et al that human plasma and urine contain substances having the same activities as thrombomodulin and the molecular weights of such substances in plasma are 63,000 and 54,000 [J. Clin. Invest., 76, 2178-2181 (1985)].
The present inventors had discovered in human urine two types of thrombin-binding substances which are different from the above-mentioned substances and having smaller molecular weights, i.e., about 39,000 and 31,000 under unreduced conditions, and filed a patent application on these substances (Japanese Patent Laid-open No. 146898/1988).
The inventors have continued further extensive studies in order to develop an advantageous process for the production, isolation, and purification of human thrombomodulin, and as a result separated from human urine two types of thrombin-binding substances [hereinafter defined as thrombin-binding substances (A) and (B)] which are different from above-mentioned previously discovered substances. The inventors confirmed that the two thrombin-binding substances are novel compounds.
The present inventors conducted further studies in order to develop an industrially advantageous process for producing and isolating these thrombin-binding substances. As a result, the inventors discovered that the same thrombin-binding substances found in urine are present in a culture broth of cells derived from human tissues and that the target substances can be separated add collected from the culture broth using simple procedures. Such findings have led to the completion of the present invention.